ARL13B regulates Sonic Hedgehog signaling from outside primary cilia

Abstract

ARL13B is a regulatory GTPase highly enriched in cilia. Complete loss of Arl13b 1 disrupts cilia architecture, protein trafficking and Sonic hedgehog signaling. To 2 determine whether ARL13B is required within cilia, we knocked in a cilia-excluded 3 variant of ARL13B (V358A) and showed it retains all known biochemical function. We 4 found that ARL13B V358A protein was expressed but could not be detected in cilia, even 5 when retrograde ciliary transport was blocked. We showed Arl13b V358A/V358A mice are 6 viable and fertile with normal Shh signal transduction. However, in contrast to wild type 7 cilia, Arl13b V358A/V358A cells displayed short cilia and lacked ciliary ARL3 and INPP5E. 8These data indicate that ARL13B's role within cilia can be uncoupled from its function 9 outside of cilia. Furthermore, these data imply that the cilia defects upon complete 10 absence of ARL13B do not underlie the alterations in Shh transduction, which is 11 unexpected given the requirement of cilia for Shh transduction. 12

The Ciliary Protein Arl13b Functions Outside of the Primary Cilium in Shh-Mediated Axon Guidance

“…Primary cilia were still present in Arl13b V358A/V358A embryos (Figure 4E). However, despite the Arl13b V358A protein being expressed (Gigante et al, 2019) and the Arl13b antibody retaining the ability to recognize the Arl13b V358A mutant protein (Figure S2), we detected no Arl13b V358A protein in primary cilia (Figure 4E).…”

“…To examine the impact of cilia-localization-deficient Arl13b on axon guidance in vivo , we generated knockin mice carrying the Arl13b V358A variant (Gigante et al, 2019). We immunostained forelimb-level E11.5 sections of wild-type and Arl13b V358A/V358A embryos for the ciliary marker IFT88.…”

“…Constant, Canada). Mouse strains used were: Arl13b hnn/hnn (MGI:3578151), Arl13b V358A (MGI:6256969, official nomenclature Arl13b em1Tc )(Gigante et al, 2019), Kif7 (MGI:4357956, Kif7tm1.2Hui ) (Cheung et al, 2009), Tulp3 −/− (MGI: 5548429 Tulp3 tm1b(EUCOMM)hmgu ) (Legue and Liem, 2019). All mouse embryos were used at embryonic day 11.5 (E11.5), as indicated in the Results section and in the figure legends.…”

Primary Cilia as a Biomarker in Mesenchymal Stem Cells Senescence: Influencing Osteoblastic Differentiation Potency Associated with Hedgehog Signaling Regulation

“…Arl13b is a regulatory GTPase highly enriched in cilia that regulates hedgehog signaling [ 32 ]; in this study, the changes in the Arl13b expression on cilia may have had an impact on the transduction of hedgehog components that regulate senescence. However, a recent study showed that Arl13b also functions outside of primary cilia [ 33 ], which is undesirable but indicates the complexity of the regulatory mechanism within cilia. We also found that in senescent P4 MSCs, suppression of the IHH, Ptch-1, Gli1, and Gli2 expression was associated with a reduction in the CDKN2A expression.…”

Ciliary and extraciliary Gpr161 pools repress hedgehog signaling in a tissue-specific manner

“…We recently conceptually simplified the complexity in Hh regulation in tissues based on genetic epistasis of Hh mutants with (Caspary et al 2007). However, such regulation does not require ciliary pools of Arl13b, suggesting Arl13b functions outside cilia in these contexts (Gigante et al 2020). Gpr161 mut1/ko embryos also show exencephaly and spina bifida, similar to Gpr161 ko (Mukhopadhyay et al retained but PKA-RI non-binding Gpr161 vl truncation mutant (Bachmann et al 2016;Pal et al 2016) is also associated with spina bifida (Matteson et al 2008), and could involve Wnt signaling defects (Li et al 2015).…”